The availability of high titer, high purity, AAV2 stocks has dramatically increased the understanding of this virus and its utility as a gene transfer vector. The rapid development of adeno-associated virus type 2 (AAV2) as a vector system is the result of improvements in the production and purification of the virions. Gene-transfer vectors based on AAV have gained popularity due to a combination of attractive features. Wild type AAV is naturally defective for replication and considered to be nonpathogenic. Recombinant AAV vectors do not contain viral genes and can transduce both mitotic and post-mitotic cells (Alexander et al., 1994; Kaplitt et al., 1994; Russell et al., 1994; McCown et al., 1996). Efficient long-term gene transfer has been reported in a number of cell types including eye, CNS, and muscle (McCown et al., 1996; Xiao et al., 1996; Flannery et al., 1997; Snyder et al., 1997). Most pre-clinical studies and current Phase I clinical trials use vectors derived from AAV2. However, vectors derived from other AAV serotypes such as AAV4 and AAV5 have proven to be more efficient in transducing certain cell types than AAV2 and could be resistant to neutralizing antibodies against AAV2 (Rutledge et al., 1998). The difference in transduction efficiencies for these serotypes appears to be the result of different mechanisms of uptake (Chiorini et al., 1997; Chiorini et al., 1999). Therefore, gene therapy vectors based on other serotypes of AAV may be useful for transducing cell types that are not efficiently transduced by AAV2 or when there are neutralizing antibodies against AAV2.
Recent advances in the production and purification of high titer rAAV vector stocks have facilitated the transition to human clinical trials. The use of affinity chromatography based on AAV2s interaction with heparin sulfate has replaced density gradient centrifugation for purification of rAAV2. Additional improvements in rAAV stock preparations include the use of deoxycholate treatment of the cell lysate, iodixanol gradient separation prior to the affinity chromatography, have resulted in high titer rAAV2 (Clark et al., 1999; Zolotukhin et al., 1999). While this method is a significant improvement over CsCl gradient centrifugation alone, the procedure results in recovery of less than 50% of the starting transducing virus. The preparation also contains a significant percentage of empty particles, which could decrease the therapeutic index of the preparation. O'Riordan et al. have developed a scalable two-step purification procedure using ion-exchange chromatography followed by sulphate affinity chromatography (O'Riordan et al., 2000). However, not all AAV serotypes bind to heparan sulfate on the cell surface. Transduction with either AAV4 or AAV5 is insensitive to heparin sulfate competition, indicating distinct interactions for these serotypes. Instead, both AAV4 and AAV5 require sialic acid on the cell surface for binding and transduction (Kaludov et al., 2001). Thus the affinity chromatography approach used for AAV2 will not be useful for purifying these isolates.
The present invention provides a simple ion exchange method for purifying different AAV serotypes (AAV2, 4 and 5) that does not rely on the affinity of the virus for heparin sulfate or sialic acid. The procedure is fast, reproducible, efficient, and yields highly purified rAAV. It is also easily amenable for large-scale production of clinical grade vector. The final rAAV stock consists primarily of full particles as analyzed by electron microscopy. HPLC-purified virus also shows an improved particle-to-infectivity ratio compared to virus purified by conventional CsCl density gradients. This new purification method will facilitate high titer, high purity production of rAAV4 and rAAV5 and will further their development as gene transfer vectors for clinical applications.